This invention relates to nucleic acid and amino acid sequences of a human beta-alanine-pyruvate aminotransferase and to the use of these sequences in the diagnosis, treatment, and prevention of diseases associated with immune disorders and cancer.
Aminotransferases catalyze the transfer of an alpha-amino group from an alpha-amino acid to an alpha-keto acid. These enzymes, also called transaminases, generally funnel alpha-amino groups from a variety of amino acids to alpha-ketoglutarate for conversion into NH4+. Aspartate aminotransferase, one of the most important of these enzymes, catalyzes the transfer of the amino group of aspartate to alpha-ketoglutarate. In most vertebrates, NH4+ is converted into urea, and is excreted.
In terrestrial vertebrates, urea is synthesized by the urea cycle. One of the nitrogen atoms of the urea synthesized by this pathway is transferred from the amino acid aspartate. The other nitrogen atom and the carbon atom are derived from NH4+ and CO2. Ornithine is the carrier of these carbon and nitrogen atoms. Other reactions of the urea cycle lead to the synthesis of arginine from ornithine, an amino acid that occurs naturally as an intermediate in arginine biosynthesis. Alanine aminotransferase, which is also prevalent in mammalian tissue, catalyzes the transfer of the amino group of alanine to alpha-ketoglutarate which producing pyruvate and glutamate. Glutamate is then oxadatively deaminated, yielding NH4+ and regenerating alpha-ketoglutarate. (See, e.g., Stryer, L., 1988 (3rd ed.). Freeman.) High levels of NH4+ are toxic to humans. The synthesis of urea in the liver is the major route of removal of NH4+, and a complete block of any of the steps of the urea cycle is usually fatal, because there is no known alternative pathway for the synthesis of urea. Inherited disorders caused by a partial block of each of the urea cycle reactions have been diagnosed. The most common condition is an elevated level of NH4+ in the blood (hyperammonemia). A nearly total deficiency of any of the urea cycle enzymes results in coma or death shortly after birth.
The discovery of a new human beta-alanine-pyruvate aminotransferase and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention of diseases associated with immune disorders and cancer.
The invention features a substantially purified polypeptide, human beta-alanine-pyruvate aminotransferase (HAPA), comprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant of HAPA having at least 90% amino acid identity to the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising a sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide identity to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide which is complementary to the polynucleotide comprising the sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for producing a polypeptide comprising a sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding HAPA under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified HAPA having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a cancer, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist to HAPA.
The invention also provides a method for treating or preventing an immune response, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist to HAPA.
The invention also provides a method for detecting a polynucleotide encoding HAPA in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding HAPA in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.